In these experiments we plan to utilize the previously derived genetic information about polA- allelic mutations and the lambda polA transducing phage system to help us determine a number of parameters of the structure of E. coli DNA polymerase I. We plan to search for polA-mutator mutants and investigate the ability of mutator polymerase species to incorporate and excise mismatched bases in DNA in vitro. The location of the active sites for pol I functions will be defined by amber mutants of the polA gene and the enzymatically active amber peptides isolated and characterized. Polypeptide fingerprinting of the mutant enzymes will be used to study the structure of the enzymatic active site. This experimental approach will be combined with a series of crosslinking studies in an attempt to define spatial relationships between major active site peptides. The conformation of the enzyme in solution will be examined by thermal denaturation experiments utilizing microcalorimetry and immunochemical studies and the growth of protein crystals for x-ray diffraction will be continued.